This application is the 35 U.S.C. xc2xa7371 filing of PCT/EP00/11787, filed Nov. 23, 2000.
The invention relates to new 3-methylene steroid derivatives with therapeutic effects and to the preparation of pharmaceutical preparations comprising a 3-alkylidene steroid.
Steroids with methylene substituents at the 3 position of the steroid skeleton and 1-2 or 4-5 double bonds are known and claimed to be useful for therapeutic use (BE 696,235 and BE 654,772, respectively). The presumed medical indication is due to anabolic, estrogenic and progestogenic actions. None of these compounds has lived up to the expectations for such use and there was no expectation for utility as medicines in other areas, although many diseases still remain unsatisfactorily treated with presently available drugs. Notably, diseases of the immune system, such as rheumatoid arthritis and autoimmune diseases are in need for better drugs. Corticosteroidal agents are of some use for the treatment of these diseases, but improvements, both with respect to efficacy and number or severity of side effects, are needed.
This invention provides for 3-methylene steroid derivatives and prodrugs thereof, which steroid derivatives have the general formula 1 
wherein
R1 is H or together with R3 forms a xcex2-epoxide or R1 is absent if there is a 5-10 or 4-5 double bond;
R2 is (C1-C5)alkyl or CF3;
R3 is xcex2H, xcex2CH3 or together with R1 forms a xcex2-epoxide or R3 is absent if there is a 5-10 double bond;
R4 is H, lower alkyl;
Y is [H, H] [OH, H], xe2x95x90O, [OH, lower alkyl], [OH, (C2-C5)alkenyl], [OH, (C2-C5)alkynyl] or (C1-C6)alkylidene, whereby said alkyl, alkenyl, alkynyl and alkylidene is optionally halogenated; or xe2x95x90NOR5, whereby R5 is H, lower alkyl.
Dotted lines represent an optional double bond.
Preferred compounds according to the invention are those wherein R4 is H and Y is [OH, H], xe2x95x90O, [OH, lower alkyl], [OH, (C2-C5)alkenyl], [OH, (C2-C5)alkynyl] or (C1-C6)alkylidene, whereby said alkyl, alkenyl, alkynyl and alkylidene optionally might be halogenated.
In this description terms have the following meaning:
A lower alkyl is a branched or unbranched alkyl group having preferably 1-6 carbon atoms, like hexyl, isobutyl, tertiary butyl, propyl, isopropyl, ethyl, and methyl. Most preferred are alkyl groups having 1-3 carbon atoms.
(C1-C5)alkyl is a branched or unbranched alkyl group having 1-5 carbon atoms, for example methyl, ethyl, isopropyl, butyl, sec-butyl, tert-butyl etc.; Preferred are alkyl groups having 1-3 carbon atoms.
(C2-C5)alkenyl is a branched or unbranched alkenyl group having 2 to 5 carbon atoms, such as ethenyl, 2-butenyl, etc. Preferred are alkenyl groups having 2-3 carbon atoms.
(C2-C5)alkynyl is a branched or unbranched alkynyl group having 2-5 carbon atoms, such as ethynyl and propynyl etc. Preferred are alkynyl groups having 2-3 carbon atoms.
(C1-C5)alkylidene is a branched or unbranched alkylidene group having 1-5 carbon atoms, such as methenyl, butylidene etc. Preferred are alkylidene groups having 2-3 carbon atoms.
Halogen is fluorine, chlorine, bromine, and iodine.
Prodrugs are compounds which are designed to form a compound of the invention in the body of a recipient for treatment. In general such prodrugs can be esters or ethers of the 17-hydroxyl function.
A preferred embodiment of this invention is a compound as described above having a 5-10 double bond. Most preferred is the compound (7xcex1,17xcex1)-7-methyl-3-methylene-19-norpregn-5(10)-en-20-yn-17-ol. Excluded from the invention are the compounds (7xcex1,17xcex2)-7xcex1-methyl-3-methylene-4-estren-17-ol, (7xcex1,17xcex1)-7-methyl-3-methylene-19-norpregn-4-ene-17-ol, (7xcex1,17xcex1)-7-methyl-3-methylene-19,21-dinorpregn-4-ene-17-ol, (7xcex1)-17-keto-7-methyl-3-methylene-4-estrene, (7xcex1,17xcex1)-7-methyl-3-methylene-19-norpregn-4-en-20-yn-17-ol, 17xcex2-hydroxy-7xcex1-methyl-3-methylene-17xcex1-propen-2-yl-4-estrene, and 17xcex2-hydroxy-7xcex1-methyl-3-methylene-17xcex1-buten-2-yl-4-estrene. The disclaimer relates to the disclosures in BE 696,235.
The epoxide compounds (R1 and R3 together form xcex2-O-) of this invention can be prepared by oxidation of a compound having the formula 2 in which the symbols have the meaning as defined above, with metachloroperoxybenzoic acid 
The 3-methylene group in a compound of the invention can be obtained by performing a Wittig reaction using methyltriphenylphosphonium bromide and potassium t-butoxide with corresponding 3-keto precursor steroids, which are characterized by formula 3, wherein Y2 is as Y, as defined above, except that it is not xe2x95x90O and the other symbols have the meaning as defined before. 
The 17-alkylidene compounds of the invention can be prepared by a Wittig reaction of a 3-ketal derivative of the 17-keto derivative according to formula 4, wherein the symbols have the meaning as defined before, with an alkyltriphenylphosphonium bromide followed by the hydrolysis of the 3-ketal function. The compounds having Yxe2x95x90H, H at the 17-position (formula 2) of the invention can be prepared by Wolff-Kishner reduction of the 17-keto moiety of the 3-ketal compound of formula 4, wherein the symbols have the meaning as defined before. The 17-oxime derivatives of the invention can be prepared by condensation of the 17-ketofunction (formula 4) with hydroxylamine derivatives. The 16-alkyl compound of the invention can be prepared by alkylation of the 16-position of the 17-keto compound (Yxe2x95x90O, formula 4). 
The above indicated reagents for transformation of the starting compounds and their ways of reacting with compounds are known in the art, but not yet applied for the group of compounds to be prepared to obtain the compounds of the invention. Starting materials can be obtained by methods described in the literature. A specifically relevant reference is Van Vliet et al. Recl.Trav.Chim.Pays-Bas; EN; 105; 4; 1986; 111-115, which text is incorporated into this description by way of reference. Notably, it is described therein that 17-alkyl-3-keto-derivatives according to formula 3, wherein Y2 is [OH, lower alkyl] and the other symbols have the meaning as defined before, can be obtained by catalytic hydrogenation, for example with PtO2/H2, of a compound according to formula 3, wherein Y2 is [OH, alkenyl] or [OH, alkynyl] and the other symbols have the meaning as defined before. Derivatives according to formula 5 can be obtained by purification from a mixture of compounds formed after the Birch reduction of the 4-en-3-one derivative according to formula 6, whereby both in formula 5 and in formula 6 the symbols have the meanings as defined before. 
A 4-en-3-one derivative according to formula 6, wherein R3 is H and the other symbols have the meanings as defined before, can be obtained from a compound according to formula 7 by double bond isomerisation (according to methods described in van Vliet et. al. 1986). 
A 4-en-3-one derivative according to formula 6, wherein R3 is methyl and the other symbols have the meanings as defined before, can be obtained according to the methods described in Grunwell et. al., Steroids Vol. 27, pages 759-771, 1976, which publication is incorporated herein by reference.
Further methods to obtain compounds according to formula 7 as starting material can be prepared as described by van Vliet et al. 1986.
The use of compounds as defined by formula 1 is for immunomodulation in mammals. A compound of this invention can in particular be used in the treatment of arthritic diseases such as rheumatoid arthritis (RA) and autoimmune diseases (AIDs) such as Sjxc3x6gren""s syndrome and systemic lupus erythematosus (SLE). It can also be used prophylactically for such diseases. Furthermore, a compound according to this invention can be used for the preparation of medicines comprising a compound of this invention as an active constituent.
The present invention also relates to a pharmaceutical composition comprising the steroid compound according to the invention, mixed with one or more pharmaceutically acceptable auxiliaries, such as described in the standard reference Gennaro et al., Remmington""s Pharmaceutical Sciences, (18th ed., Mack publishing Company, 1990, see especially Part 8: Pharmaceutical Preparations and Their Manufacture.). A mixture of one or more of the steroid compounds according to the invention and one or more pharmaceutically acceptable auxiliaries may be compressed into solid dosage units, such as pills, tablets, or be processed into capsules or suppositories. By means of pharmaceutically suitable liquids the compounds can also be applied as an injection preparation in the form of a solution, suspension, emulsion, or as a spray, e.g. nasal spray. For making dosage units, e.g. tablets, the use of conventional additives such as fillers, colorants, polymeric binders and the like is contemplated. In general any pharmaceutically acceptable additive which does not interfere with the function of the active compounds can be used. The steroid compounds of the invention may also be included in an implant, a patch, a gel, and any other preparation for sustained release. Suitable carriers with which the compositions can be administered include lactose, starch, cellulose derivatives and the like, or mixtures thereof used in suitable amounts.
In another aspect, the invention relates to the use of the steroid compound according to the invention for the manufacture of a medicament for modulation of the immune system of a mammal. More specifically, the invention relates to the use of the steroid compound according to the invention for the manufacture of a medicament for the treatment of arthritic diseases such as rheumatoid arthritis (RA) and autoimmune diseases (AIDs) such as Sjxc3x6gren""s syndrome and systemic lupus erythematosus (SLE). Such medicaments can also be prepared for prophylactic use for such diseases. The medicament is preferably made suitable, in particular for the treatment of human beings by complying to the regulations of health authorities in diverse countries. The use of the 3-methylene steroid derivatives (7xcex1,17xcex2)-7xcex1-methyl-3-methylene-4-estren-17-ol, (7xcex1,17xcex1)-7-methyl-3-methylene-19-norpregn-4-ene-17-ol, (7xcex1,17xcex1)-7-methyl-3-methylene-19,21-dinorpregn-4-ene-17-ol, (7xcex1)-17-keto-7-methyl-3-methylene-4-estrene, (7xcex1,17xcex1)-7-methyl-3-methylene-19-norpregn-4-en-20-yn-17-ol, 17xcex2-hydroxy-7xcex1-methyl-3-methylene-17xcex1-propen-2-yl-4-estrene, and 17xcex2-hydroxy-7xcex1-methyl-3-methylene-17xcex1-buten-2-yl-4-estrene is within the ambit of the present invention.
Furthermore, the invention pertains to the treatment of arthritic diseases such as rheumatoid arthritis (RA) and autoimmune diseases (AIDs) such as Sjxc3x6gren""s syndrome and systemic lupus erythematosus (SLE), comprising the administration to a patient of a compound as described previously (in a suitable pharmaceutical dosage form). The dosage amounts of the present steroids will be of the range of from 0.001 to 100 mg per administration of a subject in need of treatment. Consequently dosage units for practical use of a compound of this invention can contain an amount of active ingredient in the range of from 0.001 to 100 mg.
The immuno-modulating properties of a compound of this invention can be demonstrated and used in the following procedures.
In the delayed type hypersensitivity (DTH) procedure, the effect of a compound on the development of an immune reaction can be observed in mice (details in example 16). Briefly, the compound is administered daily and the animals are immunized subsequently with an antigen in adjuvant. After 7 days the animals are challenged locally by injecting the antigen locally (mostly in the foot) and measuring the subsequently developing local swelling in response to the deposition of the antigen. The degree of this swelling is related to the development of an immune response against the eliciting antigen. The inhibiting action of a compound of this invention on the development of this swelling can be determined, by comparing the treatment group with a placebo treated group. As a reference treatment, administration of glucocorticoids can be used.
The effect of a compound of this invention was also tested in arthritic mice.
In this procedure, mice are immunized with collagen type 11, mostly from bovine cartilage, in an adjuvant. After three weeks a booster with the same antigen is given. Approximately 7-10 days after the booster reaction edema of the joints (especially in the hind and forefeet) can be observed. This swelling increases rapidly, leading to redness, inflammation and distortion of normal function. Upon X-ray analysis disruption of normal joint architecture can be observed. Upon histological examination of these joints, severe inflammation leading to disruption of normal cartilage architecture can be observed. The degree of observable alterations can be graded; moreover, the degree of histological alterations can be graded also.
Furthermore, the effect of a compound on this invention can be observed in non-obese diabetic (NOD)-mice.
In this procedure, mice spontaneously and gradually develop auto-immune diseases with symptoms resembling those of human insulin dependent diabetes mellitus (IDDM) and Sjxc3x6gren""s syndrome. Sjxc3x6gren""s syndrome is characterized by the development of infiltrates in the salivary and lachrymal gland. In NOD mice especially the salivary gland is characterized by the gradual development of infiltrates. A compound of this invention inhibits dose-dependently the development of these infiltrates in the submandibular glands.
IDDM is characterized by the development of infiltrates in the pancreas, leading to destruction of the islets of Langerhans producing insulin. After the gradual destruction of all these islets, no insulin production is present anymore resulting in the development of IDDM.